The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit

Abstract

The influenza virus polymerase, a heterotrimer composed of three subunits, PA, PB1 and PB2, is responsible for replication and transcription of the eight separate segments of the viral RNA genome in the nuclei of infected cells. The polymerase synthesizes viral messenger RNAs using short capped primers derived from cellular transcripts by a unique 'cap-snatching' mechanism1. The PB2 subunit binds the 5′ cap of host pre-mRNAs2,3,4, which are subsequently cleaved after 10–13 nucleotides by the viral endonuclease, hitherto thought to reside in the PB2 (ref. 5) or PB1 (ref. 2) subunits. Here we describe biochemical and structural studies showing that the amino-terminal 209 residues of the PA subunit contain the endonuclease active site. We show that this domain has intrinsic RNA and DNA endonuclease activity that is strongly activated by manganese ions, matching observations reported for the endonuclease activity of the intact trimeric polymerase6,7. Furthermore, this activity is inhibited by 2,4-dioxo-4-phenylbutanoic acid, a known inhibitor of the influenza endonuclease8. The crystal structure of the domain reveals a structural core closely resembling resolvases and type II restriction endonucleases. The active site comprises a histidine and a cluster of three acidic residues, conserved in all influenza viruses, which bind two manganese ions in a configuration similar to other two-metal-dependent endonucleases. Two active site residues have previously been shown to specifically eliminate the polymerase endonuclease activity when mutated9. These results will facilitate the optimisation of endonuclease inhibitors10,11,12 as potential new anti-influenza drugs.

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Figure 1: Manganese ions stabilize PA-Nter.
Figure 2: Endonuclease activity of PA-Nter.
Figure 3: Three-dimensional structure of PA-Nter.
Figure 4: Comparison of PA-Nter with other nucleases of the PD-(D/E)XK superfamily.

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Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited with the Protein Data Bank (PDB) under accession codes 2W69 and R2W69SF.

References

  1. 1

    Plotch, S. J., Bouloy, M., Ulmanen, I. & Krug, R. M. A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription. Cell 23, 847–858 (1981)

    CAS  Article  Google Scholar 

  2. 2

    Li, M. L., Rao, P. & Krug, R. M. The active sites of the influenza cap-dependent endonuclease are on different polymerase subunits. EMBO J. 20, 2078–2086 (2001)

    CAS  Article  Google Scholar 

  3. 3

    Fechter, P. et al. Two aromatic residues in the PB2 subunit of influenza A RNA polymerase are crucial for cap binding. J. Biol. Chem. 278, 20381–20388 (2003)

    CAS  Article  Google Scholar 

  4. 4

    Guilligay, D. et al. The structural basis for cap binding by influenza virus polymerase subunit PB2. Nature Struct. Mol. Biol. 15, 500–506 (2008)

    CAS  Article  Google Scholar 

  5. 5

    Shi, L., Summers, D. F., Peng, Q. & Galarz, J. M. Influenza A virus RNA polymerase subunit PB2 is the endonuclease which cleaves host cell mRNA and functions only as the trimeric enzyme. Virology 208, 38–47 (1995)

    CAS  Article  Google Scholar 

  6. 6

    Doan, L., Handa, B., Roberts, N. A. & Klumpp, K. Metal ion catalysis of RNA cleavage by the influenza virus endonuclease. Biochemistry 38, 5612–5619 (1999)

    CAS  Article  Google Scholar 

  7. 7

    Klumpp, K., Doan, L., Roberts, N. A. & Handa, B. RNA and DNA hydrolysis are catalyzed by the influenza virus endonuclease. J. Biol. Chem. 275, 6181–6188 (2000)

    CAS  Article  Google Scholar 

  8. 8

    Tomassini, J. et al. Inhibition of cap (m7GpppXm)-dependent endonuclease of influenza virus by 4-substituted 2,4-dioxobutanoic acid compounds. Antimicrob. Agents Chemother. 38, 2827–2837 (1994)

    CAS  Article  Google Scholar 

  9. 9

    Hara, K., Schmidt, F. I., Crow, M. & Brownlee, G. G. Amino acid residues in the N-terminal region of the PA subunit of influenza A virus RNA polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion RNA promoter binding. J. Virol. 80, 7789–7798 (2006)

    CAS  Article  Google Scholar 

  10. 10

    Hastings, J. C., Selnick, H., Wolanski, B. & Tomassini, J. E. Anti-influenza virus activities of 4-substituted 2,4-dioxobutanoic acid inhibitors. Antimicrob. Agents Chemother. 40, 1304–1307 (1996)

    CAS  Article  Google Scholar 

  11. 11

    Tomassini, J. E. et al. A novel antiviral agent which inhibits the endonuclease of influenza viruses. Antimicrob. Agents Chemother. 40, 1189–1193 (1996)

    CAS  Article  Google Scholar 

  12. 12

    Parkes, K. E. et al. Use of a pharmacophore model to discover a new class of influenza endonuclease inhibitors. J. Med. Chem. 46, 1153–1164 (2003)

    CAS  Article  Google Scholar 

  13. 13

    Fodor, E. et al. A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs. J. Virol. 76, 8989–9001 (2002)

    CAS  Article  Google Scholar 

  14. 14

    Lee, M. T. et al. Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase. Nucleic Acids Res. 30, 429–438 (2002)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Honda, A., Mizumoto, K. & Ishihama, A. Minimum molecular architectures for transcription and replication of the influenza virus. Proc. Natl Acad. Sci. USA 99, 13166–13171 (2002)

    ADS  CAS  Article  Google Scholar 

  16. 16

    He, X. et al. Crystal structure of the polymerase PAC–PB1N complex from an avian influenza H5N1 virus. Nature 454, 1123–1126 (2008)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Obayashi, E. et al. The structural basis for an essential subunit interaction in influenza virus RNA polymerase. Nature 454, 1127–1131 (2008)

    ADS  CAS  Article  Google Scholar 

  18. 18

    Nishino, T., Komori, K., Tsuchiya, D., Ishino, Y. & Morikawa, K. Crystal structure of the archaeal Holliday junction resolvase Hjc and implications for DNA recognition. Structure 9, 197–204 (2001)

    CAS  Article  Google Scholar 

  19. 19

    Horton, N. C. & Perona, J. J. DNA cleavage by EcoRV endonuclease: two metal ions in three metal ion binding sites. Biochemistry 43, 6841–6857 (2004)

    CAS  Article  Google Scholar 

  20. 20

    Knizewski, L., Kinch, L. N., Grishin, N. V., Rychlewski, L. & Ginalski, K. Realm of PD-(D/E)XK nuclease superfamily revisited: detection of novel families with modified transitive meta profile searches. BMC Struct. Biol. 7, 40 (2007)

    Article  Google Scholar 

  21. 21

    Beese, L. S. & Steitz, T. A. Structural basis for the 3′-5′ exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J. 10, 25–33 (1991)

    CAS  Article  Google Scholar 

  22. 22

    Viadiu, H. & Aggarwal, A. K. The role of metals in catalysis by the restriction endonuclease BamHI. Nature Struct. Biol. 5, 910–916 (1998)

    CAS  Article  Google Scholar 

  23. 23

    Tarendeau, F. et al. Host determinant residue lysine 627 lies on the surface of a discrete, folded domain of influenza virus polymerase PB2 subunit. PLoS Pathog. 4, e1000136 (2008)

    Article  Google Scholar 

  24. 24

    Hagen, M., Chung, T. D., Butcher, J. A. & Krystal, M. Recombinant influenza virus polymerase: requirement of both 5′ and 3′ viral ends for endonuclease activity. J. Virol. 68, 1509–1515 (1994)

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Rao, P., Yuan, W. & Krug, R. M. Crucial role of CA cleavage sites in the cap-snatching mechanism for initiating viral mRNA synthesis. EMBO J. 22, 1188–1198 (2003)

    CAS  Article  Google Scholar 

  26. 26

    Gabriel, G. et al. The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host. Proc. Natl Acad. Sci. USA 102, 18590–18595 (2005)

    ADS  CAS  Article  Google Scholar 

  27. 27

    Salomon, R. et al. The polymerase complex genes contribute to the high virulence of the human H5N1 influenza virus isolate A/Vietnam/1203/04. J. Exp. Med. 203, 689–697 (2006)

    CAS  Article  Google Scholar 

  28. 28

    Ericsson, U. B., Hallberg, B. M., Detitta, G. T., Dekker, N. & Nordlund, P. Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal. Biochem. 357, 289–298 (2006)

    CAS  Article  Google Scholar 

  29. 29

    Saito, T., Owen, D. M., Jiang, F., Marcotrigiano, J. & Gale, M. Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA. Nature 454, 523–527 (2008)

    ADS  CAS  Article  Google Scholar 

  30. 30

    Baudin, F., Bach, C., Cusack, S. & Ruigrok, R. W. Structure of influenza virus RNP. I. Influenza virus nucleoprotein melts secondary structure in panhandle RNA and exposes the bases to the solvent. EMBO J. 13, 3158–3165 (1994)

    CAS  Article  Google Scholar 

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Acknowledgements

We thank the ESRF, EMBL and MRC-France for access to synchrotron facilities and the Partnership for Structural Biology for an integrated structural biology environment. The work was partially funded by the EU FLUPOL contract (SP5B-CT-2007-044263) and the ANR FLU INTERPOL contract (ANR-06-MIME-014-02). A.D. has a PhD fellowship from the MENRT. We thank J.-L. Decout for advice about the inhibitor, E. Kowalinski and C. Swale for RNA transcripts, and C. Petosa and J. Perona for a critical reading of the manuscript.

Author Contributions A.D., D.B., T.C. and A.A.M. performed the experiments, D.J.H. and F.B. supervised the search for a soluble fragment of PA, R.W.H.R. and S.C. supervised the biochemical characterization of PA-Nter, and S.C. supervised the crystallography. S.C. wrote the paper with the help of A.D., D.B., T.C. and R.W.H.R.

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Correspondence to Stephen Cusack.

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Dias, A., Bouvier, D., Crépin, T. et al. The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature 458, 914–918 (2009). https://doi.org/10.1038/nature07745

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